We propose to study Salmonella sp. interactions with macrophages. This should increase knowledge about the molecular basis of typhoid fever and other enteric diseases. Preliminary work indicates that Salmonella induce macrophage membrane ruffling and macropinocytosis. The macrophage intracellular compartment where Salmonella reside is a novel organelle we term a spacious phagosome (SP). Preliminary data suggests that the SP is a specific niche that promotes Salmonella survival within macrophages. These SP are morphologically similar to macropinosomes formed in response to growth factors and transforming agents. Macropinosomes are fluid filled organelles induced by exposure of macrophages to growth factors and transforming agents, such as macrophage colony stimulating factor (M-CSF). However, while macropinosomes induced by M-CSF fuse with lysosomes and shrink completely within 15 minutes, Salmonella containing SP persist within the cytoplasm of the cell, and have delayed acidification and fusion with lysosomes. We propose to characterize how defective in SP formation will be isolated by use of a microscopic technique that allows identification of Salmonella mutants deficient in mutations in envelope proteins for the ability to form SP. We will use these mutants and other genetic screening techniques to identify genes that promote SP formation and survival within macrophages. We will identify these mutants and other genetic screening techniques to identify genes that promote SP formation and survival within macrophages. We will identify the proteins encoded by these genes and characterize their role in pathogenesis. We will also perform experiments to extend our observations on SP formation to other mouse and human macrophages and with other serotypes of Salmonella. The contribution of macrophage signal transduction and the cytoskeleton will also be examined by performing experiments with kinase and microtubule inhibitors, and evaluation of the state of tyrosine phosphorylation of the macrophage after exposure to Salmonella.